Oligonucleotide therapeutics and uses thereof

Abstract

The present invention relates to oligonucleotide therapeutics. The oligonucleotide therapeutic agent has at least one oligonucleotide conjugated to a dodecylamine, and the oligonucleotide is conjugated to the dodecylamine at its 5'end. The oligonucleotide therapeutic agent may further include a polyethylene glycol (PEG) conjugated to the dodecylamine at the amino terminus of the dodecylamine, and may further include a peptide linker disposed between the dodecylamine and the PEG.

Description

[0001] Cross - reference to related applications

[0002] This application claims the international priority and benefits of U.S. Provisional Patent Application No. 63 / 385,258, filed on November 29, 2022, the content of which is incorporated herein by reference into the present invention.

[0003] Submission of the sequence listing in ASCII text invention format

[0004] This application includes a sequence listing submitted electronically in XML format. The XML - formatted sequence listing includes a sequence list named "P23 - 0225PCT_Sequence_Listing.xml", created on November 2, 2023, with a size of 2,875 bytes. The sequence listing included in the XML - formatted sequence listing is part of the specification and is hereby incorporated by reference in its entirety into the present invention. Technical field

[0005] The present invention relates to an oligonucleotide therapeutic agent, particularly to an oligonucleotide conjugated with dodecylamine. The oligonucleotide therapeutic agent may further include a polyethylene glycol (PEG) conjugated with the dodecylamine, and may even further include a peptide linker disposed between the dodecylamine and the PEG. Background art

[0006] According to the statistics of the World Health Organization (WHO) in 2023, more than 55 million people worldwide suffer from dementia, and approximately 10 million new cases are added each year. Among them, Alzheimer's disease (AD) is the most common neurodegenerative disease and the main cause of dementia. The disease is caused by the accumulation of β - amyloid (Aβ), the abnormal phosphorylation of Tau protein, and neuroinflammation. Most studies show that β - amyloid leads to the accumulation of plaques, further causing brain neurotoxicity, while the over - phosphorylation of tau protein forms neurofibrillary tangles (NFT), leading to irreversible neuronal cell death (Bloom, 2014).

[0007] To date, recent studies have pointed out and shown that neuroinflammation is one of the initial causes of Alzheimer's disease (Kinney et al., 2018). This neuroinflammatory phenomenon enhanced by Aβ accumulation stimulates microglia to release highly neurotoxic inflammatory factors, further promoting the occurrence of brain inflammatory responses (Lueg et al., 2015; Hansen et al., 2018). Therefore, how to inhibit and alleviate inflammatory responses, especially neuroinflammation, is extremely important for preventing neurodegenerative diseases such as dementia and Alzheimer's disease. Summary of the Invention

[0008] The present invention is at least partially based on the discovery that dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (named C12-miR) unexpectedly increases the PD-L1 expression of cells, which is completely contrary to the existing reports on the inhibitory effect of microRNA 200c-3p mimics or microRNA 200c-3p expression vectors on PD-L1 expression on cells (Anastasiadou et al., 2021; Zhang et al., 2023). The polyethylene glycolylation of C12-miR and the addition of a peptide linker (SEQ ID NO: 2) between PEG and C12-miR can further induce cells to express more PD-L1. Based on the PD-1 / PD-L1 receptor-ligand axis, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR), polyethylene glycolylated C12-miRs, and polyethylene glycolylated peptide-linked C12-miRs disclosed in the present invention can be used as oligonucleotide therapeutics to attenuate and inhibit inflammatory responses, especially neuroinflammatory responses.

[0009] Thus, in certain embodiments, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide conjugated with a dodecylamine, and the oligonucleotide is conjugated with the dodecylamine at its 5' end. In certain other embodiments, the oligonucleotide therapeutic agent may further include polyethylene glycol (PEG) conjugated to the amino terminus of the dodecylamine. In certain other embodiments, the oligonucleotide therapeutic agent may further include a peptide linker disposed between the dodecylamine and the PEG.

[0010] The present invention also provides the use or application of the oligonucleotide therapeutic agent. In certain embodiments, the oligonucleotide therapeutic agent disclosed in the present invention can be used to increase the expression of PD-L1 in a cell. In certain other embodiments, the oligonucleotide therapeutic agent disclosed in the present invention can be used to increase the expression of PD-L1 in a subject. In certain other embodiments, the oligonucleotide therapeutic agent disclosed in the present invention can be used to prevent, mitigate, inhibit, or treat an inflammatory response in a subject.

[0011] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experimentation. Such equivalents are intended to be encompassed by the following embodiments.

[0012] Embodiment 1. An oligonucleotide therapeutic agent comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, and an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine.

[0013] Embodiment 2. The oligonucleotide therapeutic agent according to Embodiment 1, wherein the oligonucleotide is a microRNA.

[0014] Embodiment 3. The oligonucleotide therapeutic agent according to Embodiment 1 or 2, wherein the oligonucleotide consists of a sequence as shown in SEQ ID NO:1.

[0015] Embodiment 4. The oligonucleotide therapeutic agent according to any one of Embodiments 1 to 3, further comprising a polyethylene glycol (PEG) conjugated to the dodecylamine at an amino group end of the dodecylamine.

[0016] Embodiment 5. The oligonucleotide therapeutic agent according to Embodiment 4, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

[0017] Embodiment 6. The oligonucleotide therapeutic agent according to any one of Embodiments 1 to 3, further comprising a peptide linker conjugated to the dodecylamine at an amino group end of the dodecylamine, and a polyethylene glycol (PEG) conjugated to the peptide linker at an amino group end of the peptide linker.

[0018] Embodiment 7. The oligonucleotide therapeutic agent according to Embodiment 6, wherein the peptide linker consists of a sequence as shown in SEQ ID NO:2.

[0019] Embodiment 8. The oligonucleotide therapeutic agent as described in Embodiment 6 or 7, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

[0020] Embodiment 9. The oligonucleotide therapeutic agent as described in any one of Embodiments 1 to 8, wherein the oligonucleotide therapeutic agent is selected from the group consisting of:

[0021] An oligonucleotide therapeutic agent composed of an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, and an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine;

[0022] An oligonucleotide therapeutic agent composed of an oligonucleotide, a dodecylamine, and a polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the dodecylamine at an amino group end of the dodecylamine; and

[0023] An oligonucleotide therapeutic agent composed of an oligonucleotide, a dodecylamine, a peptide linker, and a polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, the peptide linker is conjugated to the dodecylamine at an amino group end of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the peptide linker at an amino group end of the peptide linker.

[0024] Embodiment 10. The oligonucleotide therapeutic agent as described in any one of Embodiments 1 to 9, wherein the oligonucleotide therapeutic agent is composed of an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, and an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine.

[0025] Embodiment 11. The oligonucleotide therapeutic agent as described in any one of Embodiments 1 to 9, wherein the oligonucleotide therapeutic agent is composed of an oligonucleotide, a dodecylamine, and a polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the dodecylamine at an amino group end of the dodecylamine.

[0026] Embodiment 12. The oligonucleotide therapeutic agent according to any one of Embodiments 1 to 9, wherein the oligonucleotide therapeutic agent consists of an oligonucleotide, a dodecylamine, a peptide linker, and a polyethylene glycol (PEG), wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, the peptide linker is conjugated to the dodecylamine at an amino group end of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the peptide linker at an amino group end of the peptide linker.

[0027] Embodiment 13. The oligonucleotide therapeutic agent according to any one of Embodiments 9 to 12, wherein the oligonucleotide is a microRNA.

[0028] Embodiment 14. The oligonucleotide therapeutic agent according to any one of Embodiments 9 to 13, wherein the oligonucleotide consists of a sequence as shown in SEQ ID NO:1.

[0029] Embodiment 15. The oligonucleotide therapeutic agent according to any one of Embodiments 9 to 14, wherein the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

[0030] Embodiment 16. The oligonucleotide therapeutic agent according to any one of Embodiments 9 to 15, wherein the peptide linker consists of a sequence as shown in SEQ ID NO:2.

[0031] Embodiment 17. A composition comprising at least one oligonucleotide therapeutic agent according to any one of Embodiments 1 to 16 and a pharmaceutically acceptable carrier or excipient.

[0032] Embodiment 18. A method for increasing the expression of PD-L1 in a cell, comprising contacting the cell with an oligonucleotide therapeutic agent according to any one of Embodiments 1 to 16 or a composition according to Embodiment 17.

[0033] Embodiment 19. A method for increasing the expression of PD-L1 in a subject, comprising administering to the subject an oligonucleotide therapeutic agent according to any one of Embodiments 1 to 16 or a composition according to Embodiment 17.

[0034] Embodiment 20. A method for preventing, alleviating, inhibiting, or treating an inflammatory response in a subject, comprising administering to the subject a pharmaceutically effective amount of an oligonucleotide therapeutic agent according to any one of Embodiments 1 to 16 or a composition according to Embodiment 17.

[0035] Embodiment 21. The method according to embodiment 20, wherein the inflammatory response in the subject is a neuroinflammatory response.

[0036] Embodiment 22. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in increasing the PD-L1 expression of a cell.

[0037] Embodiment 23. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in increasing the PD-L1 expression in a subject.

[0038] Embodiment 24. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in preventing, alleviating, inhibiting, or treating an inflammatory response in a subject.

[0039] Embodiment 25. The use according to embodiment 24, wherein the inflammatory response in the subject is a neuroinflammatory response.

[0040] Embodiment 26. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in preparing a drug for increasing the PD-L1 expression of a cell.

[0041] Embodiment 27. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in preparing a drug for increasing the PD-L1 expression in a subject.

[0042] Embodiment 28. Use of the oligonucleotide therapeutic agent according to any one of embodiments 1 to 16 or the composition according to embodiment 17 in preparing a drug for preventing, alleviating, inhibiting, or treating an inflammatory response in a subject.

[0043] Embodiment 29. The use according to embodiment 28, wherein the inflammatory response in the subject is a neuroinflammatory response.

[0044] These and other aspects will become apparent from the following description of the preferred embodiments taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The drawings illustrate one or more embodiments of the present invention and, together with the written description, are used to explain the principles of the present invention. Wherever possible, the same reference numerals are used throughout the drawings to refer to the same or similar components in the embodiments.

[0046] Figure 1The figure shows a schematic diagram of the design of the oligonucleotide therapeutic agent disclosed by the present invention. miR, C12, and Linker represent microRNA, dodecylamine, and peptide linker (SEQ ID NO: 2), respectively.

[0047] Figure 2 The figure shows the characterization results of the oligonucleotide therapeutic agent prepared in Example 1, which was characterized by electrophoresis on a 15% (v / v) acrylamide gel containing 8 M urea and subsequent staining with 0.2% (w / v) methylene blue. Arrows indicate the positions of each size (7 kDa and 8 kDa) on the gel.

[0048] Figure 3 The figure shows SH-SY5Y human neuroblastoma cells ( CRL-2266) treated with phosphate-buffered saline (PBS) (as a blank control group), 2 μM C12-miR, 2 μM P.5-C12-miR, or 2 μM P.5-L-C12-miR for 48 hours.

[0049] Figure 4 The figure shows the effects of different concentrations (0.5 μM, 2 μM, and 4 μM) of C12-miR and P.5-C12-miR on the expression of PD-L1 in SH-SY5Y human neuroblastoma cells ( CRL-2266) in Example 3. The expression of PD-L1 was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. The results are presented as the mean, and the error bars represent the standard deviation. Statistical significance was calculated by Student's t-test. Compared with the blank control group, *p < 0.05, **p < 0.01, ***p < 0.001. Compared with the C12-miR group, #p < 0.05, ##p < 0.01.

[0050] Figure 5 The figure shows the effects of PEG with different sizes conjugated to C12-miR on SH-SY5Y human neuroblastoma cells ( The effect of CRL-2266 on the expression of PD-L1 was demonstrated. The expression of PD-L1 was analyzed by flow cytometry using an anti-human PD-L1 surface antibody. The results are presented as the mean, with error bars representing the standard deviation, and statistical significance was calculated by Student's t-test. *p < 0.05 compared to the positive control group (C12-miR group).

[0051] Figure 6 Shown is the size reduction of the PEGylated peptide-linked C12-miR (P.5-L-C12-miR) digested by the lysosomal enzyme cathepsin D in Example 5. P.5-L-C12-miR (control group) and P.5-L-C12-miR digested by cathepsin D were analyzed by electrophoresis in a 15% (v / v) acrylamide gel containing 8 M urea and subsequent staining with 0.2% (w / v) methylene blue. The lines indicate the size of each band. Detailed Description

[0052] The present invention is at least partially based on the discovery that dodecylamine-modified microRNA 200c-3p (SEQ ID NO:1) (C12-miR) unexpectedly increases the PD-L1 expression of cells; furthermore, the PEGylation of C12-miR and the addition of a peptide linker (SEQ ID NO:2) between PEG and C12-miR can further induce cells to express more PD-L1.

[0053] Accordingly, the present invention provides an oligonucleotide therapeutic agent having at least one oligonucleotide conjugated to dodecylamine, and the oligonucleotide is conjugated to the dodecylamine at its 5'-end. Preferably and in certain embodiments, the oligonucleotide is a microRNA. More preferably and in certain preferred embodiments, the oligonucleotide consists of a sequence as shown in SEQ ID NO:1. Preferably and in certain embodiments, the oligonucleotide therapeutic agent may further comprise polyethylene glycol (PEG) conjugated to the dodecylamine at the amino-terminus of the dodecylamine. Preferably and in certain embodiments, the oligonucleotide therapeutic agent may further comprise a peptide linker disposed between the dodecylamine and the PEG. More preferably and in certain preferred embodiments, the polyethylene glycol (PEG) is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000. Even more preferably and in certain more preferred embodiments, the polyethylene glycol (PEG) is PEG 500. More preferably and in certain preferred embodiments, the peptide linker is conjugated to the dodecylamine at the amino-terminus of the dodecylamine, and the polyethylene glycol (PEG) is conjugated to the peptide linker at the amino-terminus of the peptide linker. More preferably and in certain preferred embodiments, the peptide linker consists of a sequence as shown in SEQ ID NO:2.

[0054] The present invention also provides a composition comprising at least one oligonucleotide therapeutic agent as described in the present invention and a pharmaceutically acceptable carrier or excipient.

[0055] The present invention also provides a method for increasing the expression of PD-L1 in a cell, comprising contacting the cell with an oligonucleotide therapeutic agent as described in the present invention. The present invention also provides a method for increasing the expression of PD-L1 in a subject, comprising administering to the subject an oligonucleotide therapeutic agent as described in the present invention. The present invention also provides a method for preventing, alleviating, inhibiting, or treating an inflammatory response in a subject, comprising administering to the subject a pharmaceutically effective amount of an oligonucleotide therapeutic agent as described in the present invention.

[0056] As used in the present invention, the terms "programmed cell death protein 1", "PD-1", "CD279 (cluster of differentiation 279)" refer to a cell surface receptor protein present on certain immune cells (especially T cells), and it regulates the immune system's response to human cells by downregulating the immune system and promotes self-tolerance by inhibiting the inflammatory activity of T cells.

[0057] As used in the present invention, the terms "programmed death ligand 1", "PD-L1", "CD274 (cluster of differentiation 274)" refer to a type I transmembrane protein with a size of 40 kDa that exists on the surface of certain cells, including cancer cells and immune cells, and is capable of interacting with the inhibitory checkpoint molecule PD-1.

[0058] As used in the present invention, the term "PD-1 / PD-L1 axis" refers to an important immune regulatory pathway in the human body through the interaction between PD-1 and PD-L1. The binding of PD-1 on the surface of T cells to PD-L1 on the surface of another cell transmits an inhibitory signal, reducing the proliferation of antigen-specific T cells in lymph nodes and simultaneously reducing the apoptosis of regulatory T cells (anti-inflammatory, inhibitory T cells). The main function of the PD-1 / PD-L1 interaction is to prevent an overactive immune response that may lead to autoimmunity or excessive tissue damage. However, certain cancers also utilize it as a way to evade the immune system. Cancer cells can express PD-L1, and when they bind to PD-1 on T cells, they can effectively inhibit the ability of T cells to attack tumors. This mechanism is one of the ways for cancer cells to evade immune surveillance. In addition, the PD-1 / PD-L1 axis has been found to be an important pathway for regulating the immune system in the brain, maintaining microglial uptake of Aβ, and reducing chronic neuroinflammation. Studies have shown that the expression of PD-L1 in astrocytes and the expression of PD-1 in microglia around Aβ plaques are both upregulated, and astrocytes secrete a soluble form of PD-L1 that binds to PD-1 on microglia. The binding of PD-L1 to PD-1 increases the uptake and clearance of Aβ by microglia and inhibits the continuous expansion of Aβ, thereby inhibiting neuroinflammation (Kummer et al., 2021). Therefore, an increase in the expression of PD-L1 on nerve cells has the effect of inhibiting neuroinflammation.

[0059] As used in the present invention, the term "dodecylamine" refers to an organic compound with the chemical formula C12H27NH2 and the following chemical structural formula: Dodecylamine belongs to amines and is characterized in that a primary amine functional group (-NH2) is connected to a C12 carbon alkyl chain. As used in the present invention, the first carbon atom of dodecylamine is conjugated to the oligonucleotide at the 5' end of the oligonucleotide, and the amine group of dodecylamine is located on the twelfth carbon atom of the dodecylamine.

[0060] As used in the present invention, the term "polyethylene glycol" or "PEG" refers to a polymer compound with the chemical formula H-(O-CH2-CH2) n -OH and the following chemical structural formula: The PEG can be followed by a number representing the average molecular weight. For example, PEG 500, PEG 1000, and PEG 2000 represent PEGs with average molecular weights of 500, 1000, and 2000, respectively.

[0061] As used herein, the term "nucleotide" refers to a monomer comprising a nitrogenous base attached to a sugar phosphate, said sugar phosphate comprising a sugar such as ribose or 2'-deoxyribose attached to one or more phosphate groups. "Polynucleotide" and "nucleic acid" mean polymers comprising more than one nucleotide monomer, wherein the monomers are typically joined by sugar-phosphate bonds of a sugar-phosphate backbone. A polynucleotide need not comprise only one type of nucleotide monomer. For example, the nucleotides comprising a given polynucleotide can be ribonucleotides only, 2'-oxynucleotides only, or a combination of ribonucleotides and 2'-deoxyribonucleotides. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as nucleic acid analogs comprising one or more non-naturally occurring monomers. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which "U" replaces "T". The term "cDNA" means a DNA that is complementary or identical to an mRNA, whether in single-stranded or double-stranded form, but in which "T" replaces "U". The term "recombinant nucleic acid" means a polynucleotide or nucleic acid having sequences that are not joined together in nature. A recombinant nucleic acid can exist in the form of a vector.

[0062] As used herein, the term "oligonucleotide" refers to short DNA or RNA molecules, which typically have a length of 13-25 nucleotides. The maximum length of an oligonucleotide is about 200 nucleotide residues.

[0063] As used herein, the terms "microRNA", "miRNA" refer to short non-coding single-stranded RNA sequences composed of 18-22 nucleotides. MicroRNAs bind to complementary untranslated regions (3'-UTRs) of messenger RNA (mRNA) to regulate the expression of target genes, resulting in inhibition or degradation of the translation of target genes. Each microRNA can regulate many or even hundreds of different mRNA molecules, and multiple microRNAs can regulate the same mRNA. MicroRNAs are involved in a variety of biological functions, including development, differentiation, proliferation, apoptosis, etc.

[0064] As used in the present invention, the term "microRNA 200c-3p" or "miRNA 200c-3p" refers to a specific microRNA molecule belonging to the microRNA-200 family, which has the sequence of 5'-UAAUACUGCCGGGUAAUGAUGGA-3' (SEQ ID NO:1). Existing studies have shown that miR-200c-3p can reduce the expression of PD-L1, c-Myc, and β-catenin in ovarian cancer, indicating that miR-200c-3p can act as a tumor suppressor in epithelial ovarian cancer (Anastasiadou et al., 2021). In addition, other studies have also shown that miR-200c can inhibit the expression of PD-L1 mRNA in mouse lung tumor cells, thereby exerting an anti-tumor effect (Zhang et al., 2023).

[0065] As used in the present invention, the nomenclature for describing the peptides of the present invention follows the conventional practice, where the amine group (N-terminus) and / or 5' is located on the left, and the carboxyl group (C-terminus) and / or 3' is located on the right.

[0066] As used in the present invention, the term "peptide" refers to a molecular chain of amino acids, including L-type and D-type. If necessary, the amino acids can be modified in vivo or in vitro, such as by mannosylation, glycosylation, amidation (especially C-terminal amide), carboxylation, or phosphorylation, provided that these modifications must maintain the biological activity of the original molecule. In addition, the peptide can be part of a chimeric protein.

[0067] As used in the present invention, the term "peptide linker" refers to a short chain of amino acids (peptide fragment) used to connect or link different functional components in various biological or chemical molecules.

[0068] Functional derivatives of peptides are also included in the present invention. Functional derivatives are intended to include peptides having one or more different amino acids throughout the sequence, which have deletions, substitutions, inversions, or additions. Amino acid substitutions that are expected to substantially not alter the biological and immunological activities have been described. Amino acid replacements between related amino acids or replacements that frequently occur in evolution include Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, and Ile / Val, etc.

[0069] The peptides according to the present invention can be produced by synthetic or recombinant DNA techniques. Methods for producing synthetic peptides are known in the art.

[0070] Organic chemical methods for peptide synthesis are considered to include coupling the required amino acids by a condensation reaction, either in homogeneous phase or by means of the so-called solid phase. The condensation reaction can be carried out as follows: in the presence of a condensing agent, a compound (amino acid, peptide) having a free carboxyl group and other protected reactive groups is condensed with a compound (amino acid, peptide) having a free amino group and other protected reactive groups. A compound (amino acid, peptide) having an activated carboxyl group and free or protected other reactive groups is condensed with a compound (amino acid, peptide) having a free amino group and free or protected other reactive groups. The carboxyl group can be activated by converting it into an acyl halide, azide, acid anhydride, imidazoline, or an activated ester, such as N-hydroxysuccinimide, N-hydroxybenzotriazole, or p-nitrophenyl.

[0071] As used in the present invention, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption enhancing or delaying agents, and other excipients or additives that are physiologically compatible. In certain embodiments, the carrier is suitable for intranasal, intravenous, intramuscular, intradermal, subcutaneous, parenteral, oral, transmucosal, or transdermal administration. Depending on the route of administration, the active compound can be encapsulated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound. The use of such media and reagents for pharmaceutical active substances is known in the art.

[0072] Formulations suitable for administration in the present invention can include, which may be known to those skilled in the art: aqueous and non-aqueous solutions, antioxidants, bacteriostatic agents, buffers, solutes affecting isotonicity, preservatives, solubilizers, stabilizers, suspending agents, thickening agents, or combinations thereof.

[0073] In addition or in the alternative, formulations suitable for administration in the present invention can include, which may be known to those skilled in the art: gels, PEG such as PEG 400, propylene glycol, saline, sachets, water, other suitable liquids known in the art, or combinations thereof.

[0074] In addition or in the alternative, formulations suitable for administration in the present invention can include, which may be known to those skilled in the art: binders, buffers, calcium phosphate, cellulose, colloids such as colloidal silica, colorants, diluents, disintegrants, dyes, fillers, flavoring agents, gelatin, lactose, magnesium stearate, mannitol, microcrystalline gelatin, wetting agents, paraffin hydrocarbons, lozenges, polyethylene glycol, preservatives, sorbitol, starches such as corn starch, potato starch, or combinations thereof, stearic acid, sucrose, talc, triglycerides, or combinations thereof.

[0075] Additionally or in the alternative, formulations suitable for administration in accordance with the present invention may include, which may be known to those skilled in the art: alcohols such as benzyl alcohol or ethanol, benzalkonium chloride, buffers such as phosphate buffer, acetate buffer, citrate buffer, or combinations thereof, carboxymethyl cellulose or microcrystalline cellulose, cholesterol, glucose, fruit juices such as grapefruit juice, milk, phospholipids such as lecithin, oils such as vegetable oil, fish oil, or mineral oil, or combinations thereof; other pharmaceutically compatible carriers known in the art; or combinations thereof.

[0076] Additionally or in the alternative, formulations suitable for administration in accordance with the present invention may include, which may be known to those skilled in the art: biodegradable, such as poly(lactic-co-glycolic acid) (PLGA) polymers, and degradation products of other entities can be rapidly cleared from a biological system, or combinations thereof.

[0077] The formulations of the present invention can be administered in unit dose form, multiple dose form, or a combination thereof. They can be packaged in unit dose containers, multiple dose containers, or a combination thereof. The present invention may be present in ampoules, small capsules, capsules, granules, lozenges, powders, tablets, vials, emulsions, including but not limited to gum arabic emulsions, suspensions, or combinations thereof.

[0078] As used in the present invention, an “effective amount” or a “sufficient amount” of a substance is an amount sufficient to achieve a beneficial or desired result (including clinical results), and thus, an “effective amount” depends on the circumstances in which it is applied. In the case of administering an immunogenic composition, the effective amount is an immunogenic effective amount, which includes an amount of the immunogenic composition of the present invention sufficient to elicit an immune response. In the case of administering a pharmaceutical composition, the effective amount is a pharmaceutically effective amount, which includes an amount of the pharmaceutical composition of the present invention sufficient to maintain or produce the desired physiological result. One or more doses of an effective amount may be administered.

[0079] As used in the present invention, the term “pharmaceutically effective amount” refers to an amount capable of or sufficient to maintain or produce a desired physiological result, including, but not limited to, treating, reducing, alleviating, eliminating, inhibiting, substantially preventing, or preventing, or combinations thereof, a disease, disorder, or combinations thereof. A pharmaceutically effective amount may include one or more doses administered sequentially or simultaneously. Those skilled in the art will know to adjust the dose of the present invention to accommodate various types of formulations, including but not limited to, sustained release formulations. As used in the present invention, the term “preventive” refers to a composition capable of substantially preventing or preventing any aspect of a disease, disorder, or combinations thereof. As used in the present invention, the term “therapeutic” refers to capable of treating, reducing, stopping deterioration, slowing deterioration, beneficially altering, eliminating, or combinations thereof, any aspect of a disease, disorder, or combinations thereof.

[0080] As used herein, the term "dose" with respect to a composition refers to a measured portion of the composition that is taken (administered or received) by a subject at any given time.

[0081] As used herein, the term "subject" refers to an animal, and more particularly to a non-human mammal and a human organism. Non-human animal subjects may also include prenatal forms of the animal, such as an embryo or a fetus. Non-limiting examples of non-human animals include: horses, cows, camels, goats, sheep, dogs, cats, non-human primates, mice, rats, rabbits, hamsters, guinea pigs, pigs. In certain embodiments, the subject is a human. Human subjects may also include fetuses.

[0082] As used herein, the term "subject" refers to any subject in need of treatment, particularly a mammalian subject, such as a human.

[0083] As used herein, the terms "treat", "treating", or "treatment" include alleviating at least one of its symptoms, reducing its severity, or inhibiting its worsening. Treatment does not necessarily mean that the disease, disorder, or condition is completely cured. In order to be an effective treatment, the compositions useful in the present invention only need to reduce the severity of a disease, condition, or disorder, reduce the severity of the symptoms associated therewith, or improve the quality of life of a patient or subject.

[0084] As used herein, the terms "prevent", "preventing", or "prevention" refer to being able to substantially preclude, avoid, avert, prevent, stop, hinder, or a combination thereof the occurrence of any aspect of a disease, condition, or a combination thereof, particularly by pre-emptive action.

[0085] In certain embodiments, the oligonucleotide therapeutic agents and / or compositions of the present invention can be administered to a subject by a variety of routes of administration, including intradermal, intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, parenteral, oral, rectal, intranasal, intrapulmonary, and transdermal delivery, or topical administration to the eye, ear, skin, or mucosa. Alternatively, the antigen can optionally be administered ex vivo by direct exposure to cells, tissues, or organs derived from a subject (autologous) or another subject (allogeneic) in a biologically suitable liquid or solid carrier.

[0086] The meanings of the technical and scientific terms as described in the present invention can be clearly understood by those of ordinary skill in the art.

[0087] As used in the present invention, the terms "about", "approximately" or "substantially" when combined with a numerical value refer to plus or minus 10% of the reference value. For example, a length of about 1000 nanometers (nm) refers to a length in the range of 900 nm to 1100 nm.

[0088] As used in the present invention, the term "comprising" is open-ended, indicating that such embodiments may include additional elements. Conversely, the term "consisting of" is closed-ended, indicating that such embodiments do not include additional elements (except for trace impurities). The term "consisting essentially of" is partially closed-ended, indicating that such embodiments may also include elements that do not substantially change the basic characteristics of such embodiments.

[0089] When the applicant uses an open-ended conjunction such as "comprising" to define the invention or a part thereof, it should be readily understood that (unless otherwise stated), the specification should be construed as also using the conjunctions "consisting essentially of" or "consisting of" to describe the invention.

[0090] It should be noted that as used in the present invention and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, reference to "the polypeptide" includes one or more of the polypeptides so recited and equivalents known to those skilled in the art, and so forth. It should also be noted that the claims may be drafted to exclude any optional element. Thus, the present invention is intended to serve as a basis for precedence in the use of exclusive terms such as "solely", "only", etc. when citing claim limitations or using "negative" limitations.

[0091] In certain cases, when using conventions such as "at least one of A, B, and C", etc., generally such a construction is intended in the sense that those skilled in the art will understand the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include, but not be limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). Those skilled in the art will further understand that any disjunctive word and / or phrase that actually presents two or more alternative terms, whether in the description, claims, or drawings, should be understood as contemplating the possibility of including one of the terms, either term, or both terms. For example, the phrase "A or B" will be understood to include the possibility of "A" or "B" or "A and B".

[0092] The present invention will be further illustrated by the following examples, which are provided for demonstration rather than limitation. Those skilled in the art should understand that, based on the disclosure of the present invention, various changes can be made to the disclosed embodiments without departing from the spirit and scope of the present invention, and similar or analogous results can still be obtained.

[0093] Example

[0094] Example 1 Preparation of oligonucleotide therapeutic agent

[0095] Materials and methods

[0096] Design and preparation of oligonucleotide therapeutic agent. The schematic diagram of the design of the oligonucleotide therapeutic agent disclosed in the example is as Figure 1 shown. The preparation method of the oligonucleotide therapeutic agent is as follows. First, microRNA200c-3p (5'-UAAUACUGCCGGGUAAUGAUGGA-3'; SEQ ID NO: 1) (Genomics BioSci & Tech Co., Ltd., New Taipei City, Taiwan, China; GenScript Biotech Corporation, New Jersey, USA) was synthesized by solid-phase synthesis method, and the 5'-ribose end of the microRNA was modified with dodecylamine (C12H27N), wherein the first carbon atom of the dodecylamine was conjugated to the 5' end of the microRNA, and the amino group of the dodecylamine was located on the twelfth carbon of the dodecylamine. The obtained oligonucleotide therapeutic agent was named C12-miR.

[0097] The microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (C12-miR) was further conjugated with polyethylene glycol (PEG) of different sizes (0.5, 1, and 2 kDa) at the amino terminus of the dodecylamine. The polyethylene glycolylation of C12-miR was as follows: 4.15 nM C12-miR was mixed with 4.15 nM PEG (0.5, 1, or 2 kDa) in 2(N-morpholino)ethanesulfonic acid (MES) buffer containing 4.15 nM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and reacted at room temperature for 3 hours. Then the mixture was purified with a MicrospinTM G-25 column (Sigma-Aldrich, Missouri, USA) to obtain more oligonucleotide therapeutics of this example. C12-miR modified with PEG 500 (0.5 kDa), PEG 1000 (1 kDa), and PEG 2000 (2 kDa) were named P.5-C12-miR, P1-C12-miR, and P2-C12-miR, respectively.

[0098] In addition, the microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (C12-miR) was further modified with a peptide linker (KGDGG; SEQ ID NO:2) at the amino terminus of the dodecylamine, and the amino terminus of the peptide linker (SEQ ID NO:2) was further conjugated with a PEG 500 (0.5 kDa) to form another oligonucleotide therapeutic, named P.5-L-C12-miR. Briefly, 4.15 nM C12-miR, 4.15 nM of the peptide linker (SEQ ID NO:2), and 4.15 nM PEG 500 were mixed in MES buffer containing 4.15 nM EDC and reacted at room temperature for 3 hours. Then the mixture was purified with a MicrospinTM G-25 column to obtain the oligonucleotide therapeutic P.5-L-C12-miR.

[0099] The PEGylated C12-miR products (P.5-C12-miR, P1-C12-miR, P2-C12-miR, and P.5-L-C12-miR) were quantified by measuring the optical density at a wavelength of 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific, Massachusetts, USA). The unPEGylated and PEGylated C12-miR (C12-miR, P.5-C12-miR, P1-C12-miR, P2-C12-miR, and P.5-L-C12-miR) were characterized by electrophoresis on a 15% (v / v) acrylamide gel containing 8 M urea, followed by staining with 0.2% (w / v) methylene blue for 20 - 30 minutes.

[0100] Results

[0101] As Figure 2 shown, the unPEGylated C12-miR had an expected size of approximately 7 kDa. As the size of the conjugated PEG increased, the size of the PEGylated C12-miR (P.5-C12-miR, P1-C12-miR, and P2-C12-miR) also increased. Additionally, due to the addition of the peptide linker (SEQ ID NO:2), the PEGylated peptide-linked C12-miR (P.5-L-C12-miR) had a larger size than P.5-C12-miR.

[0102] Example 2 Biological Function Analysis of PEGylated Oligonucleotide Therapeutics With or Without Peptide Linkers

[0103] Materials and Methods

[0104] Cell treatment. SH-SY5Y human neuroblastoma cells ([[]] CRL-2266) suspended in MEM / F12K medium (1:1, v / v) were seeded onto a 96-well microplate at a density of approximately 5 x 103 cells / well. The cells were cultured at 37 °C in a 5% CO2 atmosphere for 16 hours and then treated with 2 μM of C12-miR, P.5-C12-miR, and P.5-L-C12-miR obtained in Example 1, respectively. The treated cells were then further cultured at 37 °C in a 5% CO2 atmosphere for 48 hours. Cells treated with phosphate-buffered saline (PBS) served as the blank control group.

[0105] Flow cytometry analysis. Cells were harvested and washed with PBS. The collected cells were stained with anti-human PD-L1 surface antibody (Catalog No.: 329706, Biolegend, California, USA) and incubated for 30 minutes at 4°C in the dark. Then the cells were washed twice with cold FACS buffer, resuspended in FACS buffer, and analyzed using a flow cytometer (BD LSR Fortessa TMX20, New Jersey, USA).

[0106] Statistical analysis. All data were subjected to one-tailed Student's t-test using the TTEST function of Excel software (Microsoft, Washington, USA). A p-value less than 0.05 between groups was considered statistically significant.

[0107] Results

[0108] The oligonucleotide therapeutics of the present invention induce PD-L1 expression. As Figure 3 shown, compared with the blank control group, the three tested oligonucleotide therapeutics C12-miR, P.5-C12-miR, and P.5-L-C12-miR used in this example significantly increased the PD-L1 expression in SH-SY5Y cells by 23% (p<0.01), 39% (p<0.01), and 58% (p<0.001), respectively. In particular, compared with unPEGylated C12-miR, PEGylated C12-miR (P.5-C12-miR) (p<0.05) and PEGylated peptide-linked C12-miR (P.5-L-C12-miR) (p<0.001) both significantly increased the PD-L1 expression in SH-SY5Y cells. More specifically, compared with PEGylated C12-miR (P.5-C12-miR), PEGylated peptide-linked C12-miR (P.5-L-C12-miR) also significantly increased the PD-L1 expression in SH-SY5Y cells (p<0.05).

[0109] The above results showed that microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (C12-miR) unexpectedly increased the expression of PD-L1 in cells, which was completely opposite to the reported inhibitory effects of microRNA 200c-3p mimics or microRNA 200c-3p expression vectors on the expression of PD-L1 on cells (Anastasiadou et al., 2021; Zhang et al., 2023). The above results also showed that the PEGylation of microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (P.5-C12-miR) induced more PD-L1 expression in cells than microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (C12-miR). The results further showed that adding a peptide linker (SEQ ID NO:2) between PEG and microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (P.5-L-C12-miR) induced even more PD-L1 expression in cells than microRNA 200c-3p (SEQ ID NO:1) modified with dodecylamine (C12-miR).

[0110] Example 3 Biological function analysis of PEGylated oligonucleotide therapeutics at different doses

[0111] Materials and methods

[0112] Cell treatment. SH-SY5Y human neuroblastoma cells ( CRL-2266) were also used in this example. The cells were cultured as described in Example 2, except that in this example, the cells were treated with 0.5 μM, 2 μM, and 4 μM of C12-miR or P.5-C12-miR. Cells treated with PBS served as the blank control group (i.e., treated with 0 μM of C12-miR or P.5-C12-miR).

[0113] Flow cytometry analysis. The flow cytometry analysis method was the same as described in Example 2.

[0114] Statistical analysis. The statistical analysis method was the same as described in Example 2.

[0115] Results

[0116] The oligonucleotide therapeutics of the present invention induced PD-L1 expression in a dose-dependent manner. As Figure 4As shown, compared with the blank control group (0 μM), 0.5 μM, 2 μM, and 4 μM of dodecylamine-modified microRNA 200c-3p (SEQ ID NO: 1) (C12-miR) significantly increased the PD-L1 expression in SH-SY5Y cells by 16%, 23% (p < 0.01), and 29% (p < 0.01), respectively. Similarly, compared with the blank control group (0 μM), 0.5 μM, 2 μM, and 4 μM of polyethylene glycolated C12-miR (P.5-C12-miR) significantly increased the PD-L1 expression in SH-SY5Y cells by 46% (p < 0.001), 39% (p < 0.001), and 59% (p < 0.001), respectively. In addition, 0.5 μM, 2 μM, and 4 μM of P.5-C12-miR significantly induced more PD-L1 expression in SH-SY5Y cells than 0.5 μM, 2 μM, and 4 μM of C12-miR (p < 0.05 or p < 0.01), indicating that the polyethylene glycolation of C12-miR (P.5-C12-miR) has a better effect than C12-miR in inducing cell expression of PD-L1. The above results show that both C12-miR and polyethylene glycolated C12-miR (P.5-C12-miR) increase the PD-L1 expression in neuroblastoma cells in a dose-dependent manner.

[0117] Example 4 Biological Function Analysis of Polyethylene Glycolated Oligonucleotide Therapeutics with Different Sizes of PEG

[0118] Materials and Methods

[0119] Cell treatment. SH-SY5Y human neuroblastoma cells ( CRL-2266) were also used in this example. The cells were cultured as described in Example 2, except that in this example, the cells were treated with 2 μM of C12-miR, P.5-C12-miR, P1-C12-miR, or P2-C12-miR. The cells treated with 2 μM of C12-miR were used as the positive control group.

[0120] Flow cytometry analysis. The flow cytometry analysis method was the same as described in Example 2.

[0121] Statistical analysis. The statistical analysis method was the same as described in Example 2.

[0122] Results

[0123] Polyethylene glycolated oligonucleotide therapeutics with different sizes of PEG can all induce the expression of PD-L1. As Figure 5As shown, compared with 2 μM of C12-miR, 2 μM of P.5-C12-miR, P1-C12-miR, and P2-C12-miR increased the PD-L1 expression in SH-SY5Y cells by 10% (p<0.05), 2%, and 3%, respectively. Among these PEGylated C12-miRs, C12-miR PEGylated with PEG 500 (P.5-C12-miR) significantly induced the highest PD-L1 expression in cells (p<0.05). These results indicate that PEGylation of C12-miR with different sizes of PEG has a positive effect on inducing PD-L1 expression in cells, especially PEGylation with PEG 500.

[0124] Example 5 Cleavage Analysis of Peptide Linker

[0125] Materials and Methods

[0126] Cleavage analysis. The PEGylated peptide-linked C12-miR (P.5-L-C12-miR) obtained in Example 1 was digested in vitro with the lysosomal enzyme cathepsin D. Briefly, 10 μL of P.5-L-C12-miR (4.15 nM) and 2 μL of cathepsin D (Cat. No. C8696, Sigma-Aldrich, Missouri, USA) were added to a 250 mM sodium acetate solution (pH 3.7) to a final volume of 20 μL. The reaction mixture was incubated at 37 °C for 5 hours. The reaction products were analyzed by electrophoresis at 200 V for 1 hour in a 15% (v / v) acrylamide gel containing 8 M urea. Then the gel was stained with 0.2% (w / v) methylene blue for 20 - 30 minutes and imaged using a Gel Doc EZ (Bio-Rad, California, USA) integrated with image analysis software (Image Lab, Bio-Rad). Each band stained for the nucleic acid part was analyzed and the size of the band was taken as the highest density point of each band.

[0127] Results

[0128] The peptide linker can be cleaved by cathepsin D. As Figure 6 shown, the PEGylated peptide-linked C12-miR (P.5-L-C12-miR) was cleaved by the lysosomal cathepsin D to produce smaller fragments. The results show that after the oligonucleotide therapeutic agent P.5-L-C12-miR is taken up by cells through pinocytosis / phagocytosis, the peptide linker is digested by the enzymes in lysosomes, and the dodecylamine-modified microRNA●200c-3p (SEQ ID NO:1) (C12-miR) is released into the cells to enhance the effect of C12-miR on cells.

[0129] In summary, the dodecylamine-modified microRNA 200c-3p (SEQ ID NO:1) (C12-miR) unexpectedly increased the PD-L1 expression of cells, and the polyethylene glycolylation of C12-miR could induce more PD-L1 expression. Moreover, adding a peptide linker (SEQ ID NO:2) between PEG and C12-miR could even induce cells to express more PD-L1. In addition, enzymatic cleavage of the peptide linker (SEQ ID NO:2) enabled the dodecylamine-modified microRNA 200c-3p (SEQ ID NO:1) (C12-miR) to be further released into the cells. These results indicate that the oligonucleotide therapeutic agent of the present invention can be used to prevent, attenuate, inhibit, and treat inflammatory responses, especially neuroinflammation.

[0130] Of course, many changes and modifications can be made to the above embodiments of the present invention without departing from the scope of the present invention. Therefore, for the purpose of promoting progress in the scientific and useful fields, the present invention is disclosed and is only intended to be limited by the scope defined by the claims.

[0131] References

[0132] G.S. Bloom, Amyloid-β and Tau. The trigger and bullet in Alzheimer disease pathogenesis. JAMA Neurology. 2014;71:505-508; doi:10.1001 / jamaneurol.2013.5847.

[0133] J.W. Kinney et al., Inflammation as a central mechanism in Alzheimer’s disease. Alzheimers Dement(NY). 2018;4:575-590; doi:10.1016 / j.trci.2018.06.014.

[0134] G. Lueg et al., Clinical relevance of specific T-cell activation in the blood and cerebrospinal fluid of patients with mild Alzheimer’s disease. Neurobiol. Aging. 2015; 36: 81-89; doi:10.1016 / j.neurobiolaging.2014.08.008.

[0135] D. V. Hansen et al., Microglia in Alzheimer’s disease. J. Cell. Biol. 2018; 217: 459-472; doi:10.1083 / jcb.201709069.

[0136] E. Anastasiadou et al., MiR-200c-3p contrasts PD-L1 induction by combinatorial therapies and slows proliferation of epithelial ovarian cancer through downregulation of β-catenin and c-Myc. Cells 2021; 10: 519; doi:10.3390 / cells10030519.

[0137] Q. Zhang et al., Aerosolized miR-138-5p and miR-200c targets PD-L1 for lung cancer prevention. Front. Immunol., 2023; 14: 1166951; doi:10.3389 / fimmu.2023.1166951.

[0138] Kummer et al., Microglial PD-1 stimulation by astrocytic PD-L1 suppresses neuroinflammation and Alzheimer’s desease pathology. EMBO J. 2021; 40: e108662; doi:10.15252 / embj.2021108662.

Claims

1. An oligonucleotide therapeutic agent, characterized in that, Comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, and an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine.

2. The oligonucleotide therapeutic agent according to claim 1, wherein The oligonucleotide is a microRNA.

3. The oligonucleotide therapeutic agent according to claim 1, wherein The oligonucleotide consists of a sequence as shown in SEQ ID NO:

1.

4. The oligonucleotide therapeutic agent according to claim 1, wherein Further comprising a polyethylene glycol conjugated to the dodecylamine at an amino group terminus of the dodecylamine.

5. The oligonucleotide therapeutic agent according to claim 4, wherein The polyethylene glycol is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

6. The oligonucleotide therapeutic agent according to claim 1, wherein Further comprising a peptide linker conjugated to the dodecylamine at an amino group terminus of the dodecylamine, and a polyethylene glycol conjugated to the peptide linker at an amino group terminus of the peptide linker.

7. The oligonucleotide therapeutic agent according to claim 6, wherein The peptide linker consists of a sequence as shown in SEQ ID NO:

2.

8. The oligonucleotide therapeutic agent according to claim 6, wherein The polyethylene glycol is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

9. The oligonucleotide therapeutic agent according to claim 1, wherein The oligonucleotide therapeutic agent is selected from the group consisting of: An oligonucleotide therapeutic agent comprising an oligonucleotide and a dodecylamine, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, and an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine; An oligonucleotide therapeutic agent comprising an oligonucleotide, a dodecylamine, and a polyethylene glycol, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, and the polyethylene glycol is conjugated to the dodecylamine at an amino group terminus of the dodecylamine; and An oligonucleotide therapeutic agent comprising an oligonucleotide, a dodecylamine, a peptide linker, and a polyethylene glycol, wherein the first carbon atom of the dodecylamine is conjugated to the oligonucleotide at the 5'-end of the oligonucleotide, an amino group of the dodecylamine is located at the twelfth carbon atom of the dodecylamine, the peptide linker is conjugated to the dodecylamine at an amino group terminus of the dodecylamine, and the polyethylene glycol is conjugated to the peptide linker at an amino group terminus of the peptide linker.

10. The oligonucleotide therapeutic agent according to claim 9, wherein, The oligonucleotide is a microRNA.

11. The oligonucleotide therapeutic agent according to claim 9, wherein, The oligonucleotide consists of a sequence as shown in SEQ ID NO:

1.

12. The oligonucleotide therapeutic agent according to claim 9, wherein The polyethylene glycol is selected from the group consisting of PEG 500, PEG 1000, and PEG 2000.

13. The oligonucleotide therapeutic agent according to claim 9, characterized in that, The peptide linker consists of a sequence as shown in SEQ ID NO:

2.

14. A composition comprising the oligonucleotide therapeutic agent according to claim 1 and a pharmaceutically acceptable carrier or excipient.

15. A method of increasing the expression of PD-L1 in a cell, comprising contacting the cell with the oligonucleotide therapeutic agent according to claim 1 or the composition according to claim 14.

16. Use of the oligonucleotide therapeutic agent according to claim 1 or the composition according to claim 14 for increasing the expression of PD-L1 in a cell.

17. Use of the oligonucleotide therapeutic agent according to claim 1 or the composition according to claim 14 for increasing the expression of PD-L1 in a subject.

18. Use of the oligonucleotide therapeutic agent according to claim 1 or the composition according to claim 14 for reducing the inflammatory response in a subject.

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